【作者】 孙兵；

【导师】 惠国桢； 郭礼和；

【作者基本信息】 苏州大学 ， 神经外科， 2004， 博士

【摘要】 帕金森病(Parkinson’s disease，PD)是60岁以上患者高发的一种神经系统变性疾病，其主要特征是由于黑质纹状体多巴胺(DA)神经元缺失，临床表现为运动徐缓、静止性震颤、齿轮样强直等症状。但目前仍没有有效的方法中止和延缓黑质多巴胺神经元的进行性病变。近年来，PD的治疗的研究发展迅速，方法也是多种多样，基因治疗已进入临床实验。由于表达稳定、表达时程长，干细胞介导的基因治疗已成为目前研究的热点，因此，本文对应用表达GDNF的骨髓基质干细胞治疗帕金森病进行了初步研究。 细胞介导的基因治疗的疗效取决于两个方面，即目的基因和载体细胞。根据文献报道，我们选择胶质细胞源性神经营养因子(glial cell linederived neurotrophic factor，GDNF)为目的基因。GDNF是中脑多巴胺神经元的神经营养因子，也是TGF-βs超家族中的一员，被认为是目前保护多巴胺能神经元最有效的神经生长因子，以往的实验表明GDNF长期作用下可以显著增加黑质纹状体的多巴胺能神经元的数量，并能有效改善其神经缺失症状。我们在第一部分实验中，从大鼠的胎脑组织中成功地克隆了GDNF全长cDNA序列，测序结果与Genebank完全一致。 中枢神经系统移植的载体细胞应具备以下特点：(1)细胞容易分离获取；(2)能够在体内长期存活；(3)有效地表达目的基因；(4)不会引起宿主的免疫排斥反应。目前多种供体细胞已应用于神经移植，如成纤维细胞，雪旺氏细胞，星型胶质细胞，肾上腺髓质细胞，胎脑多巴胺能神经元，都取得了一定的疗效。但仍然存在着一些问题，如成纤维细胞容易表达GDNF的骨益纂质十细胞治疗帕金森病的实验研究中文提要户生胶原导致中枢神经系统的胶质化，星型胶质细胞培养时难以控制，胎脑移植物不能整合到宿主的组织中。最近的研究表明骨髓基质干细胞 mesenehymal Stem cell，bone marrow stroma一eells，BMSC)可以分化为神经元和星型胶质细胞;BMSC可以穿越血脑屏障，与局部的组织整合，其分化方向与细胞的迁移部位有关，即表现为迁移到脑组织中的不同位置分化为不同类型的神经细胞，无排斥反应。BMSC来源丰富、取材简便、容易分离纯化培养，在一定培养条件下，可以快速扩增，因此，BMSC是神经系统疾病基因治疗理想的细胞载体。 在得到载体细胞和目的基因后，我们进行了BMSC的基因工程化和大鼠帕金森病模型的移植治疗，BMSC能够良好地被基因工程化，在体内、外均能有效产生GDNF，并且在转染后，基本保持了BMSC的生物学性状，依然具有多向分化的潜能，在神经系统内存活较长的时间;工程细胞的移植后，GDNF在脑内的表达持续了至少21天，与对照组相比，治疗组纹状体中的TH阳性细胞的数量显著增加，并且明显改善了PD鼠行为学，证明移植BMSC一GDNF是治疗PD的有效手段。更多还原

【Abstract】 Parkison’s disease (PD) is characterized by a loss of dopaminergic neurons in the substantia nigra and clinical symptoms that include bradykinesia , tremor at rest, and cogwheel rigidity. Several in vivo strategies have been used to replace dopamine in the striatum of patients and in animal model of PD. Currently one of the most promising treatments for PD is neural replacement by transplantation of engineered stem cells.Therapeutic effects of stem cell-mediated are determined by two fators, namely target gene and donor cells. In our study, glial cell line-derived neurotrophic factor is choosen treat PD. GDNF is a member of TGF- P family, which is currently considered as one of the most effective factors to protect dopaminergic neurons from degeneration. Previous data showed GDNF could increase the number of DA neurons, which resulted in the improvement of clinical symptoms of PD. Therefore , we cloned the full length cDNA sequence of rat GDNF.As for the donor cell, a candidate cell should own the following characters, such as easy to obtain and grow rapidly, survive well in vivo, express the target gene stably and long-term, not elicit the immunoreaction. A wide variety of donor cells, such as fibroblasts, embryonic ventral midbrain cells, astrocytes, embryonic stem (ES) cells, adrenal medullary cells, and carotid body cells, have been used with variable degrees of success in animal models of PD and in parkinsonian patients. However, none of these cell therapies has proven yet to be fully satisfactory due to different biological, technical and ethical reasons. For instance, gliosis indcuced by fibroblasts-produced collagen, difficulty to manipulate astrocytes in culture, ethical and legal issues raised by the cells from human fetuses, all need to be solved by finding alternative cell sources suitable for neural replacement. Marrow stromal cells (MSCs), also known as mesenchymal stem cell or colony-forming units(CFU)fibroblastic, are multipotent stem-like cells. MSCs have the advantage in neural transplantation that they can be easily obtained and expanded in culture, survive and integrate well without eliciting host immune response when transplanted into the rat brain. Furthermore, there has been increasing evidence that MSCs are capable of differentiating into both mesenchymal and nonmesenchymal lineages, especially into neurons and astrocytes in vitro and in vivo. As a result, we suggest that MSCs is suitable for neural transplantation.To investigate the therapeutic effects of GDNF-expressing BMSC on Parkinson’s Disease, we firstly transferred GDNF into BMSC. For PD model, built up by injecting 6-OHDA into the lateral SN of rat, GDNF-BMSC was stereoxically injected into the striatum of the lesion-side. 7,14,21,28 days after transplantation, APO-induced rotations were respectively observed. Expression of GDNF was detected by PCR and Western blot in mRNA, protein level. Immunical histochemistry was used to examine the variety of TH-positive cell. After transplantation, behavioral recovery was firstly observed in 7th day, which went on till 28day. Compared with control group, GDNF expression of graft group significantly increased in mRNA and protein level. Meanwhile, number of TH-cells is much more than that of control. Obviously, GDNF-BMSC can significantly improve the behavior of PD rat, which is one of the effective tools to treat PD.更多还原